Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase.

Chromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis.

Caught in the Act: Live-Cell Imaging of Plant Meiosis.

Live-cell imaging is a powerful method to obtain insights into cellular processes, particularly with respect to their dynamics. This is especially true for meiosis, where chromosomes and other cellular components such as the cytoskeleton follow an elaborate choreography over a relatively short period of time. Making these dynamics visible expands understanding of the regulation of meiosis and its underlying molecular forces. However, the analysis of meiosis by live-cell imaging is challenging; specifically in plants, a temporally resolved understanding of chromosome segregation and recombination events is lacking. Recent advances in live-cell imaging now allow the analysis of meiotic events in plants in real time. These new microscopy methods rely on the generation of reporter lines for meiotic regulators and on the establishment of ex vivo culture and imaging conditions, which stabilize the specimen and keep it alive for several hours or even days. In this review, we combine an overview of the technical aspects of live-cell imaging in plants with a summary of outstanding questions that can now be addressed to promote live-cell imaging in Arabidopsis and other plant species and stimulate ideas on the topics that can be addressed in the context of plant meiotic recombination.

A Practical Guide to Live-Cell Imaging of Meiosis in Arabidopsis.

Plants are powerful model systems to study meiosis. Our knowledge about the cytology of plant meiosis is mainly based on the analysis of fixed material. Although highly informative, this approach is limited in understanding the dynamics of meiosis. Here, we present a step-by-step instruction for a newly developed method to follow meiosis in male meiocytes of Arabidopsis in real time by confocal laser scanning microscopy. We envision that this method can be easily translated to other plant species and especially crops (e.g., Brassica, maize, and potato).

Live Cell Imaging of Male Meiosis in Arabidopsis by a Landmark-based System.

Live cell imaging has tremendously promoted our understanding of cellular and subcellular processes such as cell division. Here, we present a step-by-step protocol for a robust and easy-to-use live cell imaging approach to study male meiosis in the plant Arabidopsis thaliana as recently established. Our method relies on the concomitant analysis of two reporter genes that highlight chromosome configurations and microtubule dynamics. In combination, these reporter genes allowed the discrimination of five cellular parameters: cell shape, microtubule array, nucleus position, nucleolus position, and chromatin condensation. These parameters can adopt different states, e.g., the nucleus position can be central or lateral. Analyzing how tightly these states are associated gives rise to landmark stages that in turn allow a quantitative and qualitative dissection of meiotic progression. We envision that such an approach can also provide valuable criteria for the analysis of cell differentiation processes outside of meiosis.

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis.

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.